J. Braz. Chem. Soc. vol.21 número1

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Ar

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cle

*e-mail: [email protected]

#Present address: Instituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil

Novel 2-(R-phenyl)amino-3-(2-methylpropenyl)-[1,4]-naphthoquinones: Synthesis,

Characterization, Electrochemical Behavior and Antitumor Activity

Acácio I. Francisco,a_{Annelise Casellato,}a,#_{Amanda P. Neves,}a_{J. Walkimar de M. Carneiro,}a

Maria D. Vargas,*,a_{Lorenzo do C. Visentin,}b_{Alviclér Magalhães,}c_{Celso A. Câmara,}d_{Claudia Pessoa,}e

Letícia V. Costa-Lotufo,e_{José D. B. Marinho Filho}e_{and Manoel O. de Moraes}e

a_{Instituto de Química, Universidade Federal Fluminense, 24020-141 Niterói-RJ, Brazil} b_{Instituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil}

c_{Instituto de Química, Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil} d_{Instituto de Química, Universidade Federal Rural de Pernambuco, 52171-900 Recife-PE, Brazil} e_{Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270 Fortaleza-CE, Brazil}

Novas 2-(R-fenil)amino-3-(2-metilpropenil)-[1,4]-naftoquinonas (R = H, 4-OMe, 4-Ferrocenil, 4-Me, 3-Me, 4-I, 3-I, 4-CN, 3-CN, 4-NO₂ e 3-NO₂) derivadas do nor-lapachol

[2-hidroxi-3-(2-metilpropenil)-1,4-naftoquinona] foram obtidas em bons rendimentos. A estrutura dos compostos foi proposta com base em estudos de difração de raios-X (R = OMe, 2b), dados de RMN de 1_H

e 13_C_{e cálculos teóricos utilizando o funcional B3LYP e a base 6-311+G(2d,p). Os potenciais de}

meia-onda das aminonaftoquinonas e o deslocamento químico do hidrogênio da cadeia 3-propenil dos compostos 2a-k mostraram boa correlação com as constantes de Hammett dos substituintes presentes no anel fenileno. A avaliação da citotoxicidade evidenciou atividade antitumoral promissora para o substrato metóxi-nor-lapachol 1 e o derivado 4-ferrocenil 2c.

Novel 2-(R-phenyl)amino-3-(2-methyl-propenyl)-[1,4]-naphthoquinones (R = H, 4-OMe, 4-Ferrocenyl, 4-Me, 3-Me, 4-I, 3-I, 4-CN, 3-CN, 4-NO₂ and 3-NO₂) derived from nor-lapachol

[2-hydroxy-3-(2-methylpropenyl)-1,4-naphthoquinone] were obtained in good yields. Their structures were proposed on the basis of a single crystal X-ray diffraction study (R = OMe, 2b),

1_{H and}13_{C NMR studies and calculations using the B3LYP functional and the 6-311+G(2d,p)}

basis set. The half-wave potentials of the aminonaphthoquinones and 1_{H NMR chemical shifts of}

the 3-propenyl hydrogen in 2a-k show good correlation with the substituent Hammett constants on the phenylamino ring. The antitumor assays showed promising activity for substrate

methoxy-nor-lapachol 1 and the 4-ferrocenyl derivative 2c.

Keywords: Nor-lapachol, arylamine, aminonaphthoquinone, electrochemistry, B3LYP,

antitumor activity

Introduction

Naphthoquinones are widely distributed in nature and some of these molecules have an important role in the biochemistry of microbial energy production, by means of photosynthesis and respiratory chain.1_Compounds

containing the quinone group are known for exhibiting antitumor,2_trypanocide,3_moluscicide,4_fungicide5_and

antimalarial6_{activities. The presence of an amino group}

in quinones has led to interesting biologically active compounds.7-12

Biological activity of quinones is often related to their electrochemical behavior.13_{The ability to accept}

one or two electrons to form the corresponding radical anion (Q•−_{) or dianion (Q}2−_{) species is believed to induce}

formation of reactive oxygen species, responsible for the oxidative stress in cells.14 _{The electron-accepting capacity}

of naphthoquinones may be tuned by carbonyl position changes (1,2- x 1,4-naphthoquinones)15_{or different}

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Novel 2-(R-phenyl)amino-3-(2-methylpropenyl)-[1,4]-naphthoquinones J. Braz. Chem. Soc.

170

this type of molecules has proven to be useful.16,17_We

reported recently18_{the synthesis of a series of}

2-arylamino-1,4-naphthoquinones from 2-methoxy-1,4-naphthoquinone and arylamines in the presence of MgCl₂•6H

2O and

p-toluenesulfonic acid as catalysts. The reactions of both electron-donor and electron-attracting substituted anilines having given good yields of the respective products, we decided to investigate the analogous reactions of the methoxy-derivatives of nor-lapachol [2-hydroxy-3-(2-methyl-propenyl)-1,4-naphthoquinone]4_{and lapachol}

[2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone].19

Nor-lapachol is obtained from lapachol by the Hooker oxidation20_{and has been used as a substrate for the}

synthesis of several active compounds.9-12_{The incorporation}

of polyamines to this quinone, for example, has led to signiicant increase in the DNA topoisomerase II-α inhibition, compared to the original naphthoquinone.13,14

Furthermore, arylamino derivatives of nor-α and nor-β lapachones present potent antitumor11_{and trypanocide}

activities.9

Herein is the first report on the synthesis and characterization of the novel 2-arylamine derivatives of nor-lapachol 2a-k, including

2-(4-ferrocenyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone 2c (Figure 1) and

cytotoxic screening against the several cancer cell lines (SF-295, HCT-8, MDAMB-435 and HL-60). Because correlations between electrochemical potentials and the inhibitory activity of naphthoquinones on Epstein-Barr virus early antigen activation21_{and their cytotoxicity}22 _has

been reported, we also investigated the redox properties of these compounds by cyclic voltammetry.

Results and Discussion

Syntheses

The compounds 2a-k (Figure 2) were synthesized from

2-methoxy-3-(2-methylpropenyl)-[1,4]-naphthoquinone with various aromatic amines, in the presence of the catalysts 4-toluenesulfonic acid and MgCl₂•6H

2O in

methanol under relux.18_{The products are stable in the solid}

state and in solution. Compounds 2a, 2b were obtained

in a pure state, whereas 2c-k were puriied by column

chromatography using a mixture of ethyl acetate / hexane (1:5) as eluent. They were obtained in yields ranging from 84 to 73% and formulated on the basis of analytical and spectroscopic data (see Experimental).

The 1_{H NMR and infrared spectra of compounds}_2a_-_k

are consistent with their composition and structure. The

1_{H NMR spectra exhibit signals in the}_d _{7.5-8.2 ppm}

region as double dublets and triple dublets, attributed to the four naphthoquinone aromatic hydrogens H5-H8. Attributions were made on the basis of 1_{H x}1_H-COSY

experiments, J values and multiplicity. All expected resonances were observed in the 13_{C NMR spectra of}

compounds 2a-k. The carbonyl peaks appear around d183 and 180, and those attributed to C2 bound to the nitrogen at about d 145. The other chemical shifts are compatible with the structures proposed for these compounds. We observed that the chemical shift of H18 (Figure 2) in the

1_{H NMR spectra of}_2a_-_k_{is directly inluenced by the}

nature of the R substituent group in the phenylamino ring [5.80 (4-OMe) < dH(18) < 6.08 (4-NO2)].

X-ray structure / Theoretical calculations

The structure of 2-(4-methoxy-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone 2b was determined

by a single crystal X-ray diffraction study (Figure 3). The average C-C, C-O, C=O and C-N bond lengths are in good agreement with the literature.36_{The naphthoquinone ring}

system of 2b is approximately planar, the dihedral angle

between the naphthoquinone plane and the arylamine phenyl ring being 47.3°(1). The torsion angles around the fragments involved in the H-bond are: C(2)-N(1)-C(11)-C(12), −158.94(17)o_{, C(1)-C(2)-N(1)-C(11)}

−154.47(16)o_{and N(1)-C(2)-C(1)-O(2)}₋_{3.2(2)°. The}

planar unsaturated side chain is twisted about 54° with respect to the ring system. This is the irst reported structure of an amine derivative of nor-lapachol.

The packing of 2b involves molecules that interact

through classical and non-classical hydrogen bonds, forming a 1D ininite network along the crystallographic [100] direction (Figures 4 and 5). The carbonyl O1i_atom O O OMe + NH2 R O O NH cat1 / cat2

MeOH, reflux

R

1

Figure 1. Synthesis of the novel arylamines derived from methoxy-nor

-lapachol 1.

O

NH 2a_2b (R=H)_(R=4-OMe) 2c (R=4-Fc) 2d (R=4-Me) 2e (R=3-Me) 2f (R=4-I)

2g (R=3-I) 2h (R=4-CN) 2i (R=3-CN) 2j (R=4-NO2)

2k (R=3-NO2)

R 1 3 2 4 5 6 7 8 9

10 _H(18) 19 11 12 13 14 15 16 17 20 21

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Francisco et al. 171 Vol. 21, No. 1, 2010

makes a classical and a non-classical hydrogen bonds with H1 to the N1 atom (amino group) and with H12 to the C12 atom (C11-C16 phenyl ring) of a neighboring molecule, forming a six-membered ring [symmetry code: i = x-1, y, z]. In addition the other C=O group interacts via O2ii_with H5 to the C5 forming now a ten-membered ring. For more details of the crystal structure, see Supplementary material.

Starting from the experimental structure of 2b

(Figure 3), the geometries of 2a-2e and 2h-2i were fully

optimized with the B3LYP/6-31G(d) method.37_Energies

and molecular properties were obtained from a single-point calculation on the optimized geometries using the 6-311+G(2d,p) basis set38_{and the B3LYP functional.}39_To

conirm that the most stable conformation in the gas-phase is similar to that found in the solid state the geometry of an alternative conformation for 2b, with the 2-methylpropenyl

group bonded to position 3 of the naphthoquinone ring rotated by 180o_{was also optimized. This alternative}

conformation is 0.4 kcal mol-1_{less stable than the solid}

state conformation. The barrier for conversion between the two conformers calculated at the 6-31G(d) level is 5.5 kcal mol-1_.

Calculations of the absolute 1_{H NMR chemical shifts}

using the GIAO approach40_{confirmed that}

electron-attracting groups yield higher dH(18) values than

electron-donor groups. Calculations including solvent (chloroform) show essentially the same behavior. Interestingly, the

d_H(18) value for the alternative conformation with the 2-methylpropenyl side chain rotated by 180o_{is shifted}

highield by 1.67 ppm, compared to the same hydrogen in the most stable conformation. The fact that the experimental values are intermediate between the calculated values for the two conformations suggests that these conformations are in equilibrium in solution, although the variable temperature 1_{H NMR spectra of}_2a_{do not show broadening}

of H18 down to −90 o_{C in CD} 2Cl2.

Figure 3. View of the ORTEP plot for 2b with labeled atoms and 50% probability ellipsoids.

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172

UV-Vis spectra

The UV-Vis spectra of the compounds obtained in CHCl₃ show two absorption bands. PBE1PBE/6-311+G(2d,p) calculations indicate that the band in the 275-290 nm region can be attributed to the aromatic and quinone π-π∗

transitions and the low-energy band in the visible region between 456 and 512 nm is attributed predominantly to π phenyl-π*_{naphthoquinone transitions. Electron-donor}

substituents blue shift the latter band, whereas electron-attracting groups red shift it.

Cyclic voltammetry

The redox behavior of compounds 2a-k was evaluated

by cyclic voltammetry (CV) at room temperature in acetonitrile/Bu₄NPF₆ (0.1 mol L-1_{). The CVs were obtained}

in the potential range from +1.3 to –2.1V vs FcH/FcH+_as

internal standard (Table 1). Two quasi-reversible pairs of waves were observed for compounds 2a-i in the negative

region of the CV, which are attributed to the one-electron transfer to the naphthoquinone moiety. The redox potentials of the naphthoquinone unit are directly inluenced by the substituents in the phenylamino ring: electron-donor groups present lower E_1/2 when compared to electron-releasing groups. The complexity of the CV observed for 2j indicated

that the nitro group is also electroactive in the cathodic region studied41_{and because the reduction potentials for the}

nitro and the quinone moieties are similar, we were unable to assess the voltammetric parameters for this derivative. The data indicate that derivative 2c, R= ferrocenyl (Fc,

E_1/2 = −1.23 V) exhibit electronic properties similar to

compound 2b. This was also observed for the Fc-arylamine

derivative of lawsone.18

Good correlation of the E_1/2(1) potentials with the σ_p and σ_p−_{Hammett constants}42_{was obtained (Figures 6 and 7,}

respectively) except for the Fc group (Figure 7) for which the low σ_p− _{value (}₋_0.03)43_{has been correlated to low}

resonance contribution. The linear correlation coeficients for both plots suggest that all naphthoquinones of this series are reduced by the same mechanism. E_1/2(1) potentials do not show linear correlation with σ_m values.

Antitumor assays

The antitumor screening of compounds 2a-k was

carried out against three cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast) through an MTT assay35_{and the results, summarized in the}

Figure 5. View of the self-assembly 1D by classical and non-classical hydrogen bonds along the [100] crystallography direction. [Symmetry codes:

(i) x−1, y, z; (ii) x+1, y, z].

Table 1. Voltammetric data (V) for 2a-k vs FcH/FcH+_{as internal standard}

Compound E1/2 (1) E1/2 (2) E1/2 (1) – E1/2 (2)

2a −1.19 −1.54 0.35

2b −1.23 −1.59 0.36

2c −1.23 -

-2d −1.20 −1.58 0.38

2e −1.23 −1.50 0.27

2f −1.17 −1.57 0.40

2g −1.16 −1.48 0.32

2h −1.10 −1.47 0.37

2i −1.15 −1.53 0.38

2j - -

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Supplementary information, show that the Fc-derivative

2c and the methoxy-substrate 1 presented significant

proliferation inhibition against MDA-MB435, higher than the positive control doxorubicin (DOX). In a second set of experiments, four cell lines were used for IC₅₀ determination of previously selected compounds (1 and 2c). Only

methoxy-nor-lapachol 1 was highly active against MDA-MB435

and moderately active against HL-60 and HCT-8 cell lines (Table 2). The loss of activity of the ferrocenyl derivative 2c

may be due to decomposition during dilution and defreezing

of the solution, since this compound is slightly unstable in solution in the presence of oxygen.

Experimental

Materials and methods

Reagents and solvents were used without further puriication. Microanalyses were performed using a Perkin-Elmer CHN 2400 micro analyser at the Central Analítica, Instituto de Química, USP-São Paulo, Brazil. Melting points were obtained with a Mel-Temp II, Laboratory Devices-USA apparatus and are uncorrected. IR spectra (KBr pellets) were recorded on a FT-IR Spectrum One (Perkin Elmer) spectrophotometer. 1_{H and}13_{C NMR spectra were recorded}

with a Varian Unit Plus 300 MHz spectrometer in CDCl₃; coupling constants are reported in Hertz (Hz) and chemical shifts in parts per million (ppm) relative to internal standard Me₄Si. The hydrogen signals were attributed through coupling constant values and 1_{H ×}1_{H - COSY experiments. Electronic}

spectra were taken on a Diode Array 8452A (Hewlett Packard-HP) spectrophotometer using spectroscopic grade solvents (Tedia Brazil) in 10-3_{and 10}-4 _{mol L}-1_solutions.

Cyclic voltammograms were obtained on an Epsilon-BAS potentiostat-galvanostat from 1 × 10-3 _{mol L}-1_solutions

in chloroform containing 0.1 mol L-1_{of TBABF} 4 as

supporting electrolyte, at room temperature and under argon atmosphere. A standard three component system was used: a carbon-glassy working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode for organic media. Ferrocene was used as an internal standard (E_1/2 0.40 V vs NHE). Density functional calculations were carried out using the Gaussian03W molecular orbital package.23_{Geometries were fully optimized using the}

B3LYP functional24_{with the standard 6-31G(d) basis set}25

Solvent effects (chloroform) were estimated by single-point calculations on the gas-phase optimized geometries by mean of the continuum solvation model using the conductor-like polarisable continuum model26_{(CPCM) at the same level.}

NMR absolute chemical shifts were calculated using the GIAO (Gauge Independent Atomic Orbital) method with the B3LYP/6-311+G(2d,p) approach on the B3LYP/6-31G(d) optimized geometry. The electronic spectra were calculated using the TD (Time Dependent) methodology available in Gaussian. The PBE1PBE functional together with the 6-311+G(2d,p) basis set was employed.

Synthesis of the compounds 2a-k

Compounds 2a-k were synthesized by the same

procedure we reported recently for the synthesis of

Figure 6. Correlation between E_1/2(1) and σ_p.

Figure 7. Correlation between E_1/2(1) and σ_p−_.

Table 2. Cytotoxic activity expressed by IC₅₀ in μg mL-1_{of compounds}

1, 2c and doxorubicin (DOX), with the respective conidence intervals

Compound SF295 HCT-8 MDA-MB435 HL-60

2c > 5 > 5 > 5 > 5

1 > 5 2.53

(1.38-4.66) (0.14-0.64)0.30 (0.59-2.32)1.17

DOX 0.42

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174

2-arylamine-1,4-naphthoquinones derived from 2-hydroxy-1,4-naphthoquinone and were obtained in yields that varied from 71% (2c) to 84% (2h).18

[2-methoxy-3-(2-methylpropenyl)-[1,4]-naphthoquinone] (155 mg, 0.64 mmol) 1 in MeOH (6.00 mL) was heated under

relux in the presence of 4-toluenesulfonic acid (17.2 mg, 0.1 mmol) and MgCl₂•6H

2O (20.3 mg, 0.1 mmol) as

catalysts for 10 min. to dissolve most of 1. After addition

of the respective arylamine (0.96 mmol), the reactions were monitored by TLC (1:9 ethyl acetate/hexane) and were stopped when 1 was no more observed. The

resulting solids were iltered, washed with water and cold MeOH and dried under vacuum. The melting points and elemental analysis data are indicative of pure compounds. In contrast, the analogous reactions of methoxylapachol under the same conditions yielded the corresponding 1-aza-1,2-dihydro-5,10-anthraquinones in low yields described previously.19

2 ( P h e n y l ) a m i n o 3 ( 2 m e t h y l p r o p e n y l ) 1 , 4 -naphthoquinone(2a)

Yield: 78%. mp 131-133 o_{C. Anal. Calc. for C}

20H17NO2:

C 79.14; H 5.47; N 4.67%; found: C 79.19; H 5.65; N 4.62%. 1_{H NMR (300 MHz, CDCl}

3): d 8.13 (ddd, 1H,

J 6.51, J 1.51, J 0.49 Hz), 8.10 (ddd, 1H, J 6.51, J 1.51, J 0.49 Hz), 7.73 (td, 1H, J 6.51, J 6.51, J 1.51 Hz), 7.65 (td, 1H, J 6.51, J 6.51, J 1.51 Hz), 7.23 (br t, 1H, J 7.52 Hz), 7.07 (tt, 1H, J 7.52, J 2.05, J 1.13 Hz), 7.91 (m, 1H), 6.89 (dt, 1H, J 7.93, J 2.05, J 1.34 Hz), 5.87 (m, 1H), 1.38 (d, 3H, J 1.54 Hz), 1.24 (d, 3H, J 1.32 Hz). 13_{C NMR (75}

MHz, CDCl₃): d 188.2, 184.3, 140.2, 139.5, 137.9, 134.8, 133.6, 132.6, 130.8, 127.9, 126.7, 124.2, 120.1, 118.9, 117.0, 100.2, 25.6, 20.6. IR (KBr) ν_max/cm-1_{: 3278, 3102,}

3051, 2902, 1670, 1590, 1567.UV-Vis (CHCl₃) λ_max/nm: 290 (log ε 4.15), 463 (3.37).

2-(4-Methoxy-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2b)

21H19NO3:

3): d8.10 (m, 2H), 7.72

(td, 1H, J 7.49, J 7.49, J 1.54 Hz), 7.63 (td, 1H, J 7.70, J 1.54 Hz), 5.80 (m, 1H), 6.85 (br d, 2H, J 9.25 Hz), 6.77 (br d, 2H, J 9.25 Hz), 3.80 (s, 3H), 1.40 (d, 3H, J 1.54 Hz), 1.25 (d, 3H, J 1.32 Hz). 13_{C NMR (75 MHz, CDCl}

3):

183.7, 182.9, 156.5, 140.4, 138.8, 134.4, 133.0, 132.1, 130.8, 130.4, 126.0, 124.5, 118.4, 115.4, 114.7, 112.9, 55.4, 25.3, 20.2. IR (KBr) ν_max/cm-1_{: 3288, 3100, 3031,}

2889, 1668, 1600, 1577. UV-Vis (CHCl₃) λ_max/nm: 289 (log ε 4.20), 511 (3.45).

2-(4-Ferrocenyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2c)

Yield: 71%. mp 125-126 o_{C. Anal. Calc. for}

C₃₀H₂₅FeNO₂: C 73.93; H 5.17; N 2.87%; found: C 73.84; H 5.11; N 2.81%. 1_{H NMR (300 MHz, CDCl}

3): d8.16 (ddd,

1H, J 7.40, J 1.57, J 0.51 Hz), 8.13 (ddd, 1H, J 7.40, J 1.57, J 0.51 Hz), 7.79 (td, 1H, J 7.40, J 7.40, J 1.57 Hz), 7.69 (td, 1H, J 7.40, J 7.40, J 0.51 Hz), 7.55 (dd, 2H, J 6.12, J 2.09 Hz), 7.17 (dd, 2H, J 6.12, J 2.09 Hz), 5.82 (m, 1H), 4.61 (dd, 2H, J 2.37, J 1.98 Hz), 4.31 (dd, 2H, J 2.37, J 1.98 Hz), 4.04 (s, 5H), 1.35 (d, 3H, J 1.61 Hz), 1.24 (d, 3H, J 1.47 Hz). 13_{C NMR (75 MHz, CDCl}

3): 184.1, 181.8,

155.8, 139.4, 138.1, 131.7, 133.5, 131.3, 130.2, 129.6, 125.8, 124.3, 118.2, 114.9, 114.5, 112.3, 69.2, 68.7, 66.3, 24.9, 19.8. IR (KBr) ν_max/cm-1_{: 3281, 3098, 2880, 1670,}

1599, 1571, 1108, 994. UV-Vis (CHCl₃) λ_max/nm: 294 (log ε 4.29), 510 (3.64).

2-(4-Methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2d)

21H19NO2:

C 79.47; H 6.03; N 4.41%; found: C 79.43; H 6.01; N 4.38%.1_{H NMR (300 MHz, CDCl}

3): d8.13 (m, 2H), 7.74

(td, 1H, J 7.57, J 1.47 Hz), 7.66 (td, 1H, J 7.57, J 1.47 Hz), 7.05 (br d, 2H, J 8.06 Hz), 6.80 (br d, 2H, J 8.06 Hz), 5.85 (m, 1H), 2.34 (s, 3H), 1.40 (d, 3H J 1.47 Hz), 1.26 (d, 3H, J 1.22 Hz). 13_{C NMR (75 MHz, CDCl}

3): 182.1, 181.3,

157.4, 141.4, 137.5, 133.9, 132.9, 132.0, 131.0, 130.9, 125.6, 123.9, 119.1, 114.3, 114.1, 112.5, 49.3, 25.1, 20.8. IR (KBr) ν_max/cm-1_{: 3272, 3101, 3045, 2900, 1668, 1587,}

1565.UV-Vis (CHCl₃) λ_max/nm: 286 (log ε 4.00), 505 (3.25).

2-(3-Methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2e)

21H19NO2:

C 79.47; H 6.03; N 4.41%; found: C 80.01; H 6.03; N 4.53%.

1_{H NMR (300 MHz, CDCl}

3): d8.12 (m, 2H); 7.74 (td, 1H,

J 7.48, J 7.48, J 1.51 Hz), 7.66 (td, 1H, J 7.48, J 7.48, J 1.51 Hz), 7.70 (m, 2H); 7.06 (br d, 2H, J 8.30 Hz); 5.84 (m, 1H); 6.80 (br d, 2H, J 8.30 Hz); 2.34 (s, 3H); 1.40 (d, 1H, J 1.22 Hz); 1.26 (d, 1H, J 1.33 Hz. 13_{C NMR (75 MHz,}

CDCl₃): 181.9, 181.2, 157.3, 141.5, 137.2, 133.8, 133.5, 132.4, 131.9, 131.0, 130.5, 129.5, 125.3, 123.4, 118.9, 114.0, 113.8, 112.1, 50.3, 24.5, 20.3. IR (KBr) ν_max/cm-1_:

3277, 3106, 3049, 2906, 1669, 1588, 1570.UV-Vis (CHCl₃) λ_max/nm: 287 (log ε 4.15), 507 (3.31).

2-(4-Iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2f)

20H16INO2:

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3): d8.13 (dd, 1H, J 7.52, J 1.58

Hz), 8.10 (dd, 1H, J 7.52, J 1.39 Hz), 7.74 (td, 1H, J 7.52, J 7.52, J 1.39 Hz), 7.66 (td, 1H, J 7.52, J 7.52, J 1.58 Hz), 7.54 (br d, 2H, J 8.71 Hz), 6.65 (br d, 2H, J 8.71 Hz), 5.90 (m, 1H), 1.47 (d, 3H, J 1.53 Hz), 1.24 (d, 3H, J 1.33 Hz).

13_{C NMR (75 MHz, CDCl}

3): 184.3, 183.0, 140.3, 139.8,

137.7, 136.8, 134.9, 133.4, 132.8, 130.7, 126.5, 124.6, 118.9, 117.7, 100.3, 87.2, 25.7, 20.8. IR (KBr) ν_max/cm-1_:

3327, 3065, 2994, 1664, 1594, 1566. UV-Vis (CHCl₃) λ_max/nm: 290 (log ε 4.01), 460 (3.47).

2-(3-Iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2g)

Yield: 75%. mp 117-118 o_{C. Anal Calc. for C}

20H16INO2:

3): d8.11 (dd, 1H, J 7.61,

J 1.64 Hz), 8.08 (dd, 1H, J 7.61, J 1.45 Hz), 7.72 (td, 1H, J 7.61, J 1.45 Hz), 7.63 (td, 1H, J 7.61, J 1.64 Hz), 7.40 (m, 1H), 6.90 (m, 3H), 5.89 (m, 1H), 1.44 (d, 3H, J 1.49 Hz), 1.23 (d, 3H, J 1.28 Hz). 13_{C NMR (75 MHz, CDCl}

3):

184.1, 182.9, 140.5, 140.1, 137.3, 136.1, 134.6, 133.1, 132.4, 130.1, 126.9, 126.3, 124.3, 123.2, 118.1, 116.9, 100.1, 87.1, 25.2, 20.5. IR (KBr) ν_max/cm-1_{: 3331, 3069,}

2998, 1664, 1595, 1568.UV-Vis (CHCl₃) λ_max/nm: 284 (log ε 4.30), 456 (3.40).

2-(4-Cyano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2h)

21H16N2O2:

C 76.81; H 4.91; N 8.53%; found: C 76.79; H 4.87; N 8.50%.

3): d 8.14 (dd, 1H, J 1.59, J 7.46

Hz), 8.12 (dd, 1H, J 1.49, J 7.46 Hz), 7.77 (td, 1H, J 7.46, J 7.46, J 1.49 Hz), 7.70 (td, 1H, J 7.46 Hz, J 7.46, J 1.59 Hz), 7.52 (br d, 2H, J 8.58 Hz), 6.90 (br d, 2H, J 8.58 Hz), 6.03 (m, 1H), 1.50 (d, 3H, J 1.48 Hz), 1.24 (d, 3H, J 1.27 Hz). 13_C

NMR (75 MHz, CDCl₃): 184.4, 182.8, 141.9, 141.4, 138.6, 135.1, 133.2, 132.0, 130.7, 127.0, 126.7, 121.7, 120.3, 119.2, 118.8, 106.1, 100.3, 25.9, 21.0. IR (KBr) ν_max/cm-1_{: 3282,}

2-(3-Cyano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2i)

Yield: 82%. mp 173-174 o_{C. Anal Calc. for C}

21H16N2O2:

3): d8.15 (ddd, 1H, J

5.25 Hz, J 1.60 Hz, J 0.46 Hz), 8.12 (ddd, 1H, J 5.25 Hz, J 1.60 Hz, J 0.46 Hz), 7.76 (td, 1H, J 5.25, J 5.25, J 1.60 Hz), 7.69 (td, 1H, J 5.25, J 5.25, J 1.60 Hz), 7.34 (m, 2H), 7.11 (m, 2H), 5.95 (m, 1H), 1.47 (d, 3H, J 1.37 Hz), 1.25 (d, 3H, J 1.14 Hz). 13_{C NMR (75 MHz, CDCl}

3): 183.7,

182.4, 140.8, 138.7, 138.2, 134.7, 132.8, 132.7, 130.2, 128.3, 126.7, 126.5, 126.2, 126.0, 124.9, 118.4, 118.1, 111.5, 25.3, 20.5). IR (KBr) ν_max/cm-1_{: 3281, 3081, 2970,}

2925, 2218, 1661, 1591, 1564. UV-Vis (CHCl₃) λ_max/nm: 284 (log ε 4.02), 475 (3.19).

2-(4-Nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2j)

20H16N2O4:

3): d 8.14 (m, 4H), 7.87

(br s, 1H), 7.78 (td, 1H J 7.59, J 7.59, J 1.47 Hz), 7.71 (td, 1H, J 7.59, J 1.57 Hz), 6.91 (br d, 2H, J 9.08 Hz), 6.08 (m, 1H), 1.27 (d, 3H, J 1.17 Hz), 1.53 (d, 3H, J 1.43 Hz).

13_{C NMR (75 MHz, CDCl}

3): 184.0, 182.3, 143.4, 142.4,

141.4, 138.0, 134.7, 132.7, 132.9, 130.7, 126.6, 123.6, 120.4, 120.8, 118.3, 99.8, 25.6, 20.7. IR (KBr) ν_max/cm-1_:

3283, 3084, 2974, 2911, 1593, 1663, 1500, 1332. UV-Vis (CHCl₃) λ_max/nm: 281 (log ε 4.37), 469 (3.63).

2-(3-Nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2k)

20H16N2O4:

3): d8.15 (ddd, 1H, J

7.57, J 1.46, J 0.49 Hz), 8.13 (ddd, 1H, J 7.57, J 1.46, J 0.49 Hz), 7.91 (ddd, 1H, J 8.06, J 1.93, J 0.98 Hz), 7.82 (br s, 1H), 7.77 (td, 1H, J 7.57, J 7.57, J 1.46 Hz), 7.69 (td, 1H, J 7.57, J 1.46 Hz), 7.67 (br s, 1H), 7.40 (br t, 1H, J 8.06 Hz), 7.21 (dd, 1H, J 8.06, J 1.95 Hz), 6.01 (m, 1H), 1.43 (d, 3H, J 1.46 Hz), 1.24 (d, 3H, J 0.98 Hz). 13_{C NMR}

(75 MHz, CDCl₃): 183.8, 182.4, 147.3, 141.1, 138.6, 134.7, 132.8, 132.7, 130.2, 128.1, 127.1, 126.5, 126.2, 118.7, 118.4, 117.9, 116.3, 25.4, 20.6). IR (KBr) ν_max/cm-1_{: 3279,}

X-ray crystallography

The X-ray diffraction data for 2b were collected at

295 K from a Enraf-Nonius Kappa-CCD27_{diffractometer}

with graphite monochromatized Mo K_α radiation. The cell parameters were obtained and reined using PHICHI28_and

EvalCCD29_{programs. Intensities for (}₁_{) were corrected by}

Lorentz polarization and absorption with the SADABS30

program. The structure was solved by SHELXS-97 Direct Methods,29_{and reined with SHELXL-97,}32_contained

within the WinGX-32 crystallography program.33_The

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176

their respective C atoms, with U_iso(H) = 1.2Ueq(Csp2_{). H}

atoms bonded to C atoms in the methyl group were located geometrically and with the C-H distances ixed at 0.96Å for Csp3 _{and with U}

iso(H) = 1.5Ueq(Csp3). The positional

parameters of atom H1 bonded to N1 was obtained from a Fourier difference map and reined freely with an isotropic displacement parameter; the distance for N1-H1 is 0.87(2). X-ray data are listed in Table 3 and ORTEP-334_{for Windows}

was used to draw the Figures.

Antitumor assays

The compounds (1-5 mg mL-1_{) were tested for}

cytotoxic activity against four cancer cell lines: SF-295 (Central Nervous System), HCT-8 (colon), MDAMB-435 (breast) and HL-60 (human leukemia). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol L-1_{glutamine, 100 U mL}-1

penicillin, and 100 µg mL-1_{streptomycin at 37}o_{C with 5%}

CO₂. Each compound was dissolved in DMSO and diluted with water to obtain a concentration of 1 mg mL-1_{. They}

were incubated with the cells for 72 h. The negative control received the same amount of DMSO (0.5% in the highest concentration). Doxorubicin (0.1-0.58 µg mL-1_{) was used}

as a positive control. The cell viability was determined by

reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-phenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as described by Mosmann.35

Conclusions

The eleven novel aminonaphthoquinones 2a-k, obtained

from methoxy-nor-lapachol and various arylamines, were synthesized in good yields and showed interesting electrochemical behavior due to the nature of the substituents in the phenylamino ring, presenting a good correlation with Hammett parameter, which conirms that the reaction with electron-donor or electron-attracting groups follow a single mechanism. Unfortunately, because the arylamine derivatives of nor-lapachol were not active against the tested tumor cells, correlation between structure, electrochemical data and antitumor activity could not be attempted.

Supplementary Information

Supplementary data associated with this paper are available free of charge at http://jbcs.sbq.org.br, as a PDF ile and contain the results of the theoretical calculations, crystallographic data, NMR spectra (1_{H and APT), cyclic}

voltammograms and antitumor assays of compounds 2a-k.

Crystallographic data for the structural analysis of the three complexes have been deposited with the Cambridge Crystallographic Data Center, CCDC 734112. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1233336 033; e-mail: [email protected]).

Acknowledgments

The authors thank Prof. A. V. Pinto and Dr. M. C. F. R. Pinto for providing nor-lapachol and the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for inancial support. Pronex-FAPERJ (grant number E-26/171.512/2006) is acknowledged. J. W. D. Carneiro, M. D. Vargas, A. P. Neves and C. A. Camara are recipients of CNPq research fellowships. A. I. Francisco and A. Casellato were beneited with CAPES fellowships. We also thank the X-ray diffraction laboratory (LDRX) of Universidade Federal Fluminense for data collection.

References

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Table 3. Crystal data and structure reinement for compound 2b

Formula C₂₁H₁₉O₃N

Formula weight 333.37

Crystal system, space group Triclinic, P-1 Crystal size / mm 0.30 × 0.20 × 0.15 Unit cell dimensions

a, b ,c /Å α, β, γ /o

a =7.8709(16) b = 9.3748(19) c = 12.117(2) α = 86.11(3)° β = 81.60(3)° γ = 78.26(3)°

Volume / Å3 _865.3(3)

Z, Calculated density / (g cm-3₎ _{2 / 1.279}

T / K 295

Absorption coeficient /mm-1 _0.086

θ range 3.99 to 25.00

F(000) 352

Relections collected / unique 10324 / 3023

Rint 0.0358

Max. and min. transmission 0.9873 and 0.9748 Data/ restraints / parameters 3023 / 0 / 232

S 1.027

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Received: August 11, 2009

Web Release Date: November 12, 2009

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*e-mail: *e-mail: [email protected]

#Present address: Instituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil

Novel 2-(R-phenyl)amino-3-(2-methylpropenyl)-[1,4]-naphthoquinones: Synthesis,

Characterization, Electrochemical Behavior and Antitumor Activity

Acácio I. Francisco,a_{Annelise Casellato,}a#_{Amanda P. Neves,}a_{J. Walkimar de M. Carneiro,}a

Maria D. Vargas,*,a_{Lorenzo do C. Visentin,}b_{Alviclér Magalhães,}c_{Celso A. Câmara,}d_{Claudia Pessoa,}e

Letícia V. Costa-Lotufo,e_{José D. B. Marinho Filho}e_{and Manoel O. de Moraes}e

a_{Instituto de Química, Universidade Federal Fluminense, 24020-141 Niterói-RJ, Brazil} b_{Instituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil}

c_{Instituto de Química, Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil} d_{Instituto de Química, Universidade Federal Rural de Pernambuco, 52171-900 Recife-PE, Brazil} e_{Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270 Fortaleza-CE, Brazil}

1. Theoretical Calculations

Starting from the experimental structure for 2b (4-Me),

the geometries of 2a-2e and 2h-2i were fully optimized

with the B3LYP/6-31G(d) method. Energies and molecular properties were obtained from a single-point calculation using the 6-311+G(2d,p) basis set and the B3LYP functional. To conirm that the most stable conformation in the gas-phase is similar to that found in the solid state the

geometry of an alternative conformation for 2b, with the

2-methyl-propenyl group bonded to the position 3 of the

naphthoquinone ring rotated by 180o_{was also optimized.}

This alternative conformation was found 0.4 kcal mol-1

less stable than the solid state conformation. The barrier for conversion between the two conformers calculated at

the 6-31G(d) level is 5.5 kcal mol-1_.

The orientation of the 2-phenylene ring is stabilized by an intramolecular hydrogen bond with one carbonyl group of the naphthoquinone ring and was, therefore, not further investigated. NMR absolute chemical shifts were calculated using the GIAO (Gauge Independent Atomic Orbital) method with the B3LYP/6-311+G(2d,p) approach on the B3LYP/6-31G(d) optimized geometry. Absolute energies and GIAO nuclear magnetic shielding tensors for

H(18) are given in Table S1. Interestingly, the d_H18 value for

the alternative conformation (with the 2-methyl-propenyl

group rotated by 180o_{, entry}_2b_{-4 in Table S1) is 1.67 ppm}

shifted highield as compared to the same hydrogen in the

most stable conformation. Calculation for 2b on a geometry

with the methoxy group rotated by 90o_(entry_2b_{-2 in Table}

S1) yielded essentially the same d_H18 value as that obtained

for the most stable conformation. To verify the effect of the solvent on the chemical shifts some calculations were repeated with the solvent chloroform, using the CPCM approach. These calculations revealed that the solvent has only marginal inluence on the relative chemical shifts.

The electronic spectra were calculated using the TD (Time Dependent) methodology available in Gaussian. The PBE1PBE functional together with the 6-311+G(2d,p) basis set was employed. All calculations were carried out with the G03W package of molecular orbital calculation.

2. Crystallographic Data

2.1. Computing details

Data collection: COLLECT (Nonius, 1998); cell

reinement: PHICHI (Duisenberg, 2000); data reduction:

EVALCCD (Duisenberg, 2003); program(s) used to solve

structure: SHELXS97 (Sheldrick, 1997); program(s) used

to reine structure: SHELXL97 (Sheldrick, 1997); molecular

graphics: ORTEP-3 for Windows (Farrugia, 1997); software

used to prepare materialfor publication: WinGX publication

routines (Farrugia, 1999).

2.2. Experiment X-ray diffraction

The X-ray diffraction data for (2b) were collected at

295 K from a Enraf-Nonius Kappa-CCD diffractometer

with graphite-monochromatized Mo K_α radiation. The cell

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S2

EvalCCD programs. Intensities for (2b) were corrected

by Lorentz polarization and absorption with the SADABS

program. The structure was solved by SHELXS-97 Direct

Methods, and reined with SHELXL-97, contained within

the WinGX-32 crystallography program. The positional

parameters of the H atoms bonded to C atoms in the phenyl rings were obtained geometrically, with the C-H

distances ixed in 0.93Å for Csp2_{, and reined as riding on}

their respective C atoms, with U_iso(H) = 1.2Ueq(Csp2_{). H}

atoms bonded to C atoms in the methyl group were located geometrically and with the C-H distances ixed at 0.96Å

for Csp3 _{and with}_U

iso(H) = 1.5Ueq(Csp3). The positional

parameters of H(1) bonded to N(1) was obtained from a Fourier difference map and reined freely with an isotropic displacement parameter; the distance for N(1)-H(1) is 0.87(2). X-ray data are listed in Table S2.

Crystallographic data for the structural analysis of

compound 2b have been deposited with the Cambridge

Crystallographic Data Center, CCDC 734112. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1233336 033; e-mail: [email protected]).

The packing of 2b involves molecules that interact intra-

and intermolecularly through classical and non-classic hydrogen bonds, forming a 1D ininite network along the [100] crystallographic direction (Figures S2 and S3). The

naphthoquinone carbonyl O1i_interacts_via_{classical and}

non-classic hydrogen bonds with H(1) to N(1) (imine group) and with H(12) to C(12) [C(11)-C(16) phenyl ring] of a neighboring molecule forming a six membered

ring [symmetry code: i = x−1, y, z]. In addition the other

carbonyl group interacts via O2ii_{with H(5) to C(5) forming}

a ten membered ring in association with N(1)-H(1)…_O(1).

The H-bond geometric parameters are listed in Table S4. Table S5 gathers the atomic coordinates and equivalent isotropic displacement parameters.

Table S1. Absolute energies (Hartree) and GIAO nuclear magnetic shielding tensors (ppm) for compounds 2a-2e and 2h-2i. All calculations were carried out on a optimized B3LYP/6-31G(d) geometry

Absolute energies / hartree GIAO nuclear magnetic shielding tensors / ppm

Derivative B3LYP/6-31+G(d,p) B3LYP/6-311+G(2d,p) B3LYP/6-31+G(d,p)a _{B3LYP/6-311+G(2d,p)}b

2a (H) -977.62466 - 24.64

-2b-1 (4-OMe) -1092.15228 -1092.40283 24.64 24.97

2b-2 (4-OMe)c _-1092.14589 _- _24.67

-2b-3 (4-OMe)d _-1092.16808 _- _24.64

-2b-4 (4-OMe)e _-1092.16687 _- _26.31

-2l (3-OMe) -1092.15346 -1092.40399 24.62 24.89

2c (4-Fc) -2627.17389 -2627.55599 24.66 24.79

2m (3-Fc) - -2627.55562 - 24.85

2d (4-Me) -1016.94536 -1017.17288 24.60 24.86

2e (3-Me) - -1017.17322 - 24.81

2h-1 (4-CN) -1069.87032 -1070.11410 24.50 24.79

2h-2 (4-CN)d _-1069.89020 _- _24.46

-2i-1 (3-CN) -1069.86851 -1070.11232 24.54 24.75

2i-2 (3-CN)d _-1069.88800 _- _24.49

-a_{At this level, the absolute GIAO nuclear magnetic shielding of TMS is 32.09 ppm.}b_{At this level, the absolute GIAO nuclear magnetic shielding of TMS}

is 31.82 ppm. c _{Conformation with the methoxy group rotated by 90}o_.d_{Calculation with inclusion of solvent (CPCM, chloroform). The absolute GIAO} nuclear magnetic shielding of TMS at this level is 31.63 ppm e_{Alternative conformation, with the 2-methyl-propenyl group rotated by 180}o_{, including} solvent effect (chloroform).

Figure S1. View of the ORTEP plot for 2b with labeled atoms and 50%

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Francisco et al. S3 Vol. 21, No. 1, 2010

Figure S3. View of the self-assembly 1D by classical and non classical hydrogen bonds along the [100] crystallography direction. [Symmetry codes: (i) x−1, y, z; (ii) x+1, y, z].

Table S2. Crystal data and structure reinement for 2b.

Formula C₂₁H₁₉O₃N

formula weight 333.37

crystal system, space group Triclinic, P-1

crystal size / mm 0.30 x 0.20 x 0.15

a /Å 7.8709(16)

b/Å 9.3748(19)

c/Å 12.117(2)

α / (o₎ _86.11(3)

β / (o₎ _81.60(3)

γ / (o₎ _78.26(3)

V / Å3 _865.3(3)

Z 2

T / K 295

ρ_calc. / (g cm-3₎ _1.279

μ / mm-1 _0.086

2θ range 3.99 to 25.00

F(000) 352

Relections collected 10324

Relections unique 3023

R_int 0.0358

Max. and min. transmission 0.9873 and 0.9748

datab_{/restraints/parameters} _{3023 / 0 / 232}

Sb,c _1.027

R₁a,d _0.0430

wR₂b,e _0.1031

largest difference peak and hole / (e Å-3₎ _{0.153 and -0.180}

a_F

o2≥ 2σ(Fo2). bFo2≥ 3σ(Fo2). cS = [Σw(Fo2 – Fc2)2/(n – p)]1/2. dR1 = Σ||Fo| - |F_c||Σ|F_o|. e_wR

2 = [Σw(Fo2 –Fc2)2/Σw(Fo4)]1/2

Table S3. Geometric parameters for molecule 2b (Å, °)

Bonds distances N(1)-C(11) 1.424(2) N(1)-C(2) 1.367(2) O(1)-C(4) 1.234(2) O(2)-C(1) 1.223(2) O(3)-C(14) 1.378(2) O(3)-C(17) 1.423(2) C(3)-C(18) 1.480(2) C(19)-C(18) 1.328(3) C(19)-C(20) 1.502(3) C(19)-C(21) 1.510(3) Bond angles C(2)-N(1)-C(11) 129.43(15) C(14)-O(3)-C(17) 116.93(16) O(1)-C(4)-C(3) 120.81(16) O(1)-C(4)-C(10) 119.19(16) O(2)-C(1)-C(9) 121.52(16) O(2)-C(1)-C(2) 119.54(16) C(19)-C(18)-C(3) 126.38(17) C(20)-C(19)-C(21) 115.62(18)

Table S4. Geometric parameters for non-classical H bonds for molecule (2b) (Å, °)

D-H···A D-H H···A D···A ∠D-H···A

N(1)—H(1) ···O(2) 0.87 (2) 2.18 (2) 2.620 (2) 111.2 (18)

N(1)—H(1) ···O(1)i _{0.87 (2)} _{2.46 (2)} _{3.241 (2) 150.2 (18)}

C(12)—H(12)···O(1)i _0.93 _2.58 _{3.246 (2)} ₁₂₉

C(5)—H(5) ···O(2)ii _0.93 _2.39 _{3.304 (2)} ₁₆₉

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S4

Table S5. Atomic coordinates (× 104_{) and equivalent isotropic displacement parameters (A}2_{× 10}3_{) for (}_2b_{). U(eq) is deined as one third of the trace of} the orthogonalized Uij tensor

x y z U(eq)

C(19) −974(2) −1627(2) 5732(2) 43(1)

C(20) −2387(3) −494(2) 5299(2) 52(1)

C(21) −31(3) −2776(3) 4922(2) 70(1)

O(1) 1355(2) −120(2) 7804(1) 54(1)

O(2) −5563(2) 1227(2) 9030(1) 54(1)

C(2) −3178(2) −323(2) 8018(1) 33(1)

N(1) −4432(2) −1001(2) 7743(1) 38(1)

C(1) −3973(2) 916(2) 8784(1) 36(1)

C(9) −2800(2) 1709(2) 9233(1) 35(1)

O(3) −4254(2) −6772(1) 6747(1) 57(1)

C(10) −984(2) 1312(2) 8931(1) 35(1)

C(4) −239(2) 152(2) 8106(2) 36(1)

C(3) −1409(2) −604(2) 7634(1) 34(1)

x y z U(eq)

C(11) −4275(2) −2456(2) 7413(1) 34(1)

C(12) −5544(2) −2751(2) 6832(2) 40(1)

C(18) −559(2) −1641(2) 6757(2) 39(1)

C(16) −2999(2) −3610(2) 7728(2) 40(1)

C(8) −3498(2) 2840(2) 9955(2) 44(1)

C(14) −4310(2) −5325(2) 6920(2) 39(1)

C(15) −3008(2) −5025(2) 7473(2) 43(1)

C(5) 112(2) 2050(2) 9372(2) 44(1)

C(13) −5574(2) −4176(2) 6589(2) 43(1)

C(6) −601(3) 3166(2) 10106(2) 51(1)

C(7) −2398(3) 3565(2) 10390(2) 51(1)

C(17) −5788(3) −7133(2) 6440(2) 61(1)

3.

1

_{H NMR and APT Spectra (CDCl}

3

) of Compounds 2a-k

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Figure S5. APT spectrumof 2-(phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2a).

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S6

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Figure S10. 1_{H NMR}_spectrum_{of 2-(4-methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2d_).

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S8

Figure S12. 1_{H NMR}_spectrum_{of 2-(3-methylphenylene)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2e_).

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Figure S14. 1_{H NMR}_spectrum_{of 2-(4-iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2f_).

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S10

Figure S16. 1_{H NMR}_spectrum_{of 2-(3-iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2g_).

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Figure S18. 1_{H NMR}_spectrum_{of 2-(4-ciano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2h_).

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S12

Figure S20. 1_{H NMR}_spectrum_{of 2-(3-ciano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2i_).

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Figure S22.1_{H NMR}_spectrum_{of 2-(4-nitro-phenylene)-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2j_).

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S14

Figure S24. 1_{H NMR}_{spectrum of 2-(3-nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (}_2k_).

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Figure S25. APT spectrum of 2-(3-nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2k).

Figure S26. VT 1_{H NMR spectra of compound}_2a_{(range: 25}o_{C to}₋₉₀o_{C) in CD} 2Cl2.

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S16

4. Cyclic Voltammograms (100mV) of Compounds 2a-k

Figure S27. CV of 2-(phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone(2a).

Figure S28. CV of 2-(4-methoxy-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2b).

Figure S29. CV of 2-(4-ferrocenyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2c).

Figure S30. CV of 2-(4-methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2d).

Figure S31. CV of 2-(3-methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2e).

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Figure S33. CV of 2-(3-iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2g).

Figure S34. CV of 2-(4-ciano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2h).

Figure S35. CV of 2-(3-ciano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2i).

Figure S36. CV of 2-(4-nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2j).

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S18

5. Antitumor Assays

The antitumor screening of compounds 2a-k was

carried out against three cancer cell lines: SF-295 (central

nervous system), HCT-8 (colon), MDAMB-435 (breast)

through an MTT assayref_{and the results are summarized}

in Table S6.

Table S6. Screening of the cytotoxic activity (growth inhibition %) of compounds 2a-k and 1

Compounds SF295 GI% HCT-8 GI% MDA-MB435 GI%

Average SD Average SD Average SD

2a (H) 68.20 4.80 73.75 1.68 54.20 5.00

2b (4-OMe) -11.01 11.89 -2.95 13.01 24.54 9.15

2c (4-Fc) 2.57 7.43 5.45 6.24 102.31 1.09

2d (4-Me) 12.20 3.71 12.33 2.76 17.21 3.58

2e (3-Me) 58.59 0.40 71.52 1.01 43.86 3.54

2f (4-I) -4.93 23.88 10.29 2.13 -7.63 3.42

2g (3-I) 2.49 2.20 15.92 4.49 25.14 13.42

2h (4-CN) -5.79 0.56 2.65 0.84 10.72 0.93

2i (3-CN) -12.56 2.48 6.73 6.36 -20.51 4.35

2j (4-NO₂) 8.78 0.90 17.00 1.44 -18.20 8.24

2k (3-NO₂) -7.40 4.20 17.03 2.02 4.01 7.68

1 41.09 15.08 27.01 3.12 106.05 0.16

DOX 96.19 0.63 94.85 3.99 95.38 2.23